Canine Lyme disease is caused by infection with Borrelia species (spp.) spirochetes, including primarily B. burgdorferi sensu stricto (ss) in the United States and B. burgdorferi ss, B. garinii, and B. afzelii in Europe (Baranton et al., Int. J. Sys. Bacteriol. 1992, 42:378-383; Hovius et al., J. Clin. Microbiol. 2000, 38:2611-2621). The spirochetes are transmitted as the infected Ixodes spp. ticks obtain a blood meal, and the resulting infection in canines results in clinical signs ranging from subclinical synovitis to acute arthritis and arthralgia (Jacobson et al., Semin. Vet Med. Surg. 1996, 11:172-182; Summers et al, J. Comp. Path. 2005, 133:1-13). Importantly, the incidence of canine Lyme disease cases continues to increase annually coincident with increased numbers of human cases (Haninkova et al., Emerg. Infect. Dis. 2006, 12:604-610).
The antibodies produced in response to infection with Borrelia spp. have two distinct functions, but heretofore, both responses could be ineffective at eliminating sequestered spirochetes from a mammalian host. A number of explanations have been postulated for this defect in the normal immune response to natural infection, including antigenic variation (Schwan, Biochem. Soc. Trans. 2003, 31:108-112; Tokarz et al., Infect. Immun. 2004, 72:5419-5432) host mimicry (Barbour et al. Microbiol. Rev. 1986, 50:38-400), and intracellular localization (Ma et al., Infect. Immun. 1991, 59:671-678).
The most common humoral immune response is the production of non-specific binding/opsonizing (coating) antibodies that “mark” the spirochete for ingestion by phagocytic cells. Unfortunately, opsonizing antibodies are induced by several proteins common to other microorganisms (viz. 41 kDa proteins that comprise bacterial flagella), making their value for vaccination-induced antibody-mediated immunity, at best, questionable.
A second common immune response is the production of borreliacidal (lethal) antibodies. In contrast to opsonizing antibodies, borreliacidal antibodies recognize epitopes on only a few Borrelia spp. proteins. After binding to the specific target on the spirochete, the borreliacidal antibodies most commonly induce complement to form a membrane attack complex that kills the organism without the necessity of scavenging by phagocytic cells.
The canine Lyme disease bacterins presently employed in vaccines were developed to provide protection by inducing OspA borreliacidal antibodies (Hsien-Chu et al., JAVMA 1992, 201:403-411; Ma et al., Vaccine 1996, 14:1366-1374; Wikle et al., Intern. J. Appt Res. Vet. Med. 2006, 4:23-28; Straubinger et al., Vaccine 2001, 20:181-193) that kill the OspA-expressing spirochetes in the infected ticks as the parasites procure a bloodmeal (Fikrig et al., Proc. Natl. Acad. Sci. USA 1992, 89:5418-5421). Straubinger et al., (Vaccine 2002, 20:181-193) has reported that a whole cell vaccine induced significantly higher titers of borreliacidal antibodies than a recombinant OspA. Although such vaccines have been reasonably successful, vaccination failures have been reported (Levy et al. JAVMA 1993, 202:1834-1838; Ma et al., Vaccine 1996, 14:1366-1374; Schutzer et al., N. Engl. J. Med. 1997, 337:794-795).
It is now understood that the OspA borreliacidal antibodies generated often fail to sterilize feeding ticks, because the antibodies only recognize B. burgdorferi ss (Jobe et al., J. Clin. Microbiol. 1994, 32:618-622; Lovrich et al., Infect. Immun. 1995, 63:2113-2119) that are expressing OspA, and the ticks are commonly infected with B. burgdorferi ss spirochetes that are not expressing OspA (Fikrig et al., Infect. Immun. 1995, 63:1658-1662; Ohnishi et al., Proc. Natl. Acad. Sci. 2001, 98:670-675). In addition, the ticks are commonly also infected with other pathogenic Borrelia spp. including B. afzelli and B. garinii (Ornstein et al., J. Clin. Microbiol. 2001, 39:1294-298), while the OspA antibodies are genospecies specific (Lovrich et al., Infect. Immun. 1995, 63:2113-2119). Moreover, the ‘window of opportunity’ for protection by OspA borreliacidal antibodies is limited even when the spirochetes are susceptible, because the expression of OspA, which mediates attachment to the tick midgut (Pal et al., J. Clin. Invest. 2000, 106:561-569), is down-regulated shortly after the infected tick begins feeding (Schwan et al., Proc. Natl. Aced. Sci. USA 1995, 92:2909-2913).
B. burgdorferi ss OspC is another potential target for borreliacidal antibody-mediated immunity (Rousselle et al., J. Infect. Dis. 1998, 178:733-741). This protein appears to have an epitope that is responsible for inducing borreliacidal antibodies and is conserved among the pathogenic Borrelia spp. (Lovrich et al., Clin. Diagn. Lab. Immunol. 2005, 12:746-751). Although the specific function of the OspC protein remains unknown, it has been suggested that OspC expression is required for infection of mammals, but not for infection of ticks (Grimm et al. 2004, Proc. Natl. Acad. Sci. 101(9):3142-3147). In any event, Lyme disease spirochetes express OspC shortly after the tick begins feeding (Schwan et al., Proc. Natl. Acad. Sci. USA 1995, 92:2909-2913) and must continue to express OspC in order to establish an infection in mammals (Stewart et al., Infect. Immun. 2006, 74:3547-3553, Tilly et al., Infect. Immun. 2006, 74:3554-3564). Therefore, the “window of effectiveness” of the OspC borreliacidal antibodies is increased significantly compared to OspA borreliacidal antibodies.
It has been shown that the OspC protein can induce protective borreliacidal antibodies (Rousselle et al., J. Infect. Dis. 1998, 178:733-741, Ikushima et al., FEMS Immunol. Med. Microbiol. 2000, 29:15-21), but some previous “mapping” studies have localized the epitopes to highly heterogeneous regions of the protein (Buckles et al., Clin. Vacc. Immunol. 2006, 13:1162-1165). Therefore, borreliacidal OspC antibodies raised against these regions would only provide antibody-mediated immunity against a small number of Borrelia spp. isolates. Lovrich et al., (Clin. Diagn. Lab. Immuno. 2005, 12:746-751) identified an OspC borreliacidal antibody epitope within the C-terminal 7 amino acids (OspC7) of the protein. Most significantly, the epitope is conserved among the pathogenic Borrelia spp. However, traditional laboratory B. burgdorferi ss isolates that express OspA (i.e., contain the ospA/ospB operon) cannot be manipulated in the laboratory to also induce significant levels of OspC borreliacidal antibodies without significantly impairing their ability to induce OspA borreliacidal antibodies. Moreover, vaccinating with killed traditional laboratory B. burgdorferi ss isolates that express OspA does not induce borreliacidal OspC antibodies (Schwan et al., 1995, Proc. Natl. Acad. Sci. USA, 92:2909-2913, Obonyo et al., 1999, J. Clin. Microbiol., 37:2137-2141).
Callister et al., (U.S. Pat. Nos. 6,210,676 and 6,464,985, incorporated by reference herein) have suggested employing an immunogenic polypeptide fragment of OspC, alone or in combination with an OspA polypeptide, to prepare a vaccine to protect humans and other mammals against Lyme disease. Livey et al. (U.S. Pat. No. 6,872,550, incorporated by reference herein) also proposed a vaccine for immunizing against Lyme disease prepared from a combination of recombinant OspA, OspB, and OspC proteins. However, to date, no recombinant protein vaccine has been shown to be an improvement over the vaccines that are currently marketed. Therefore, there remains a longstanding need in the art for an improved vaccine to protect mammals, and especially canines, from Lyme disease.
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